Isolation, identification, characterization, and screening of rhizospheric bacteria for herbicidal activity
The consistent application of agrochemical herbicides has been reported to impact negatively on human health, environment, and food safety, and facilitated the emergence of weed resistances. Rhizosphere bacteria (RB) of different crops were screened for antagonism against Amaranthus hybridus L. (pigweed) and Echinochloa crus-galli (L.) Beauv. (barnyard grass) using necrosis assay technique. A total of eight rhizosphere bacterial isolates (B1–B8) produced different degrees of leaf necrosis on target weeds with isolate B2 manifesting the most significant necrotic activity. The rhizospheric bacterium (B2) with the highest necrotic activity was identified using 16S rRNA sequencing technique and further investigated. Molecular, morphological, and biochemical characterizations confirmed B2 isolate to be Pseudomonas aeruginosa. On isolation with ethyl acetate, separation, defatting, purification, and flash chromatography, seven different fractions (fraction 1–fraction 7) were obtained out of which fraction 4 showed the highest necrotic activity in necrosis assay experiment. Preparative HPLC of fraction 4 resulted in a pure compound that completely inhibited seed germination and seedling development of pigweed and barnyard grass but remained non-antagonistic to other tested soil fungi used in this study. The result obtained from this present study consequently confirmed the antagonistic behavior of rhizosphere-inhabiting P. aeruginosa to the target weeds and qualified the suitability of bacterium as good alternative source of bioherbicide. Potential herbicidal formulation from P. aeruginosa will help reduce crop loss due to weed challenges while offering a partial solution to the use of agrochemicals and food security.
Keywords16S rRNA gene DNA sequencing Deleterious rhizosphere bacteria Phylogenetic tree PHPLC and bioherbicide
Many microbes are potential alternative sources to synthetic herbicides and demonstrate a naturally specialized protection to a range of crops (Flores-Vargas and O’Hara 2006; Kennedy and Stubbs 2007; Harding and Raizada 2015). Synthetic herbicides which may include glyphosate (Hamid et al. 2011), dicamba (Mark et al. 2007), carfentrazone-ethyl (Baghestani et al. 2007), foramsulfuron (Nurse et al. 2007), isoproturon (Muhammad et al. 2012), 2,4-d sodium salt (Makhan et al. 2013), and organic herbicides such as vinegar, clove oil (Brainard et al. 2013), acetic acid (Ivany 2010), corn gluten meal (Johnson et al. 2013), d-limonene, and pelargonic acid (Allen and Randall 2014; Abouziena et al. 2009) are used in global weed control management. Weed management paradigm in recent decades is changing from conventional practices to the use of environment-friendly classical biological and bioherbicidal weed control approaches in forestry, horticulture, and crop production (Javaid and Adrees 2009; Javaid 2010; Javaid et al. 2010, 2011; Javaid and Ali 2011). The shift according to Xiaoya and Mengmeng (2016) was connected to the residual buildup of human health compromising chemicals in the environment combined with the development of weed resistance to synthetic herbicides. While the application of bioherbicides in both horticulture and crop farming is growing, its widespread use in complementing the conventional methods in an integrated weed management system is environmentally, biologically, technically, and commercially limited (Kremer 2005).
Members of fluorescent pseudomonads are commonly free-living, gram-negative microorganisms found in soils, freshwater, and marine environments. They predominate the plant rhizosphere due to the exudation of organic acids, sugars, and amino acids by the plant roots (Lugtenberg and Dekkers 1999). This group of bacteria which includes Pseudomonas, Rhizobacter, Azobacter, Rugamonas, Serpens, and Mesophilobacter has the capacity to grow on simple media compared to media with a large variety of low molecular weight organic compounds. Additionally, they intimately interact with plant roots where they equally invigorate as well as maximize plant nutrient uptake and consequently promote plant overall healthiness, protection, and soil fertility (Kloepper et al. 1980).
The occurrence of weed-associated bacteria that biologically attack weeds has been reported (Kremer et al. 1990). Several studies described the use of agricultural weed-associated fluorescent rhizobacteria as bioherbicides against dicotyledons (Charudattan 1991; Cattelan et al. 1999) and grassy weeds in cereal crop fields (Gurusiddaiah et al. 1994; Xiaoya and Mengmeng 2016). The use of microorganisms as bioherbicides has distinctively lowered the impact of the weed population with valued attraction over conventional methods that have repercussions on the natural ecosystems (Van Driesche et al. 2010). Crump et al. (1999) reported high degree of specificity for target weeds, little or zero effect on non-target crops or humans, and the effective management of herbicide-resistant weed populations as some of the advantages of bioherbicide broadcast applications in weed control. Additionally, bioherbicides may have both pre- and post-emergence effects on weed species (Bolton and Elliott 1989). One group of microorganisms largely overlooked as biocontrol agents of weeds is the deleterious rhizobacteria (DRB) that colonize plant root surfaces and possess the innate ability to inhibit root and shoot development of weeds (Schippers et al. 1987; Kennedy et al. 1991).
Weeds are common pernicious, fast-growing plants in most parts of Africa especially in the tropical and sub-tropical ecosystems of the world. They made up a major fraction of pest challenges in many African farms, plantations, and orchards where they significantly smothered economic crops while simultaneously threatening yields (Takim and Amodu 2013). Echinochloa crus-galli (L.) Beauv. (barnyard grass) and Amaranthus hybridus L. (pigweed) are some of the major weeds threatening economic crop productions. The production of tomatoes (Adigun and Lagoke 2003), maize (Chikoye et al. 2004), sugar cane (Takim et al. 2015), cowpea (Usman et al. 2002; Adigun et al. 2014), bambara groundnut (Asiwe and Kutu 2007), and rice (Ekeleme et al. 2007; Adeosun et al. 2009; FAOSTAT 2013) in many Nigerian farms and orchards may be depleted by 50–86% by unchecked weed invasion (Adigun et al. 2014). Weeds are noted for their seed resilience or long-term survival in soil, rapid population establishment, ability to occupy conveniently human disturbed areas, regenerative characteristics, reproductive capacity, adaptation to spread, development of resistant phenotypes, and abundant seed production (Poston et al. 2000; Heap 2005).
The study aimed to increase the biological sources of bioherbicides by screening rhizospheric bacteria that have potential herbicidal activity and to characterize such using molecular techniques as the basis for bioherbicide formulations.
Materials and methods
Isolation and culture preparation of rhizospheric bacterial
Details on the location and plant species from where the isolates used in the present investigation were isolated
Piper nigrum L. (pepper)
Ogbomosho, Oyo state
Triticum aestivum L.(wheat)
Ilorin, Kwara state
Zea mays L. (maize)
Ogbomosho, Oyo state
Sorghum bicolor (L.) Moench. (Sorghum)
Ilorin, Kwara state
Oryza sativa L. (Oryza)
Ilorin, Kwara state
Psidium guajava L. (guava)
Ogbomosho, Oyo state
Solanum lycopersicum L. (tomatoes)
Ilorin, Kwara state
Carica papaya L. (pawpaw)
Ogbomosho, Oyo state
Bioherbicidal screening of isolates using leaf necrosis assay
The pure colonies of the isolated RB (B1–B8) were each prepared in Mueller Hinton broth and incubated in a rotary shaker (140 rpm) for 12 h at 27 °C. Detached leaves from the target weeds (E. crus-galli and A. hybridus) were surface-sterilized with ethanol, were rinsed until all traces of ethanol are removed, and were each treated with 1 μl of the cell-free filtrate per one leaf surface. This was later transferred to previously labeled petri plates (in triplicates) containing moistened filter paper and cotton ball. The plates were incubated at 25 °C for 7 days and monitored daily for signs of necrotic lesions.
Biochemical and morphological characterization
The biochemical characterization of all the isolates was done according to Cappuccino and Sherman (1992). The tests conducted were gelatin liquefaction, lipid hydrolysis, starch hydrolysis, casein hydrolysis, indole production, hydrogen sulfide test, oxidase test, catalase test, urease test, denitrification, acid and gas production, and arginine hydrolysis. The bacterial isolates were structurally studied using a light microscope and oil immersion objective (×100), gram stain, and fluorescent tests.
Isolation of genomic DNA
The rhizopheric bacterial isolate B2 with the highest degree of herbicidal activity was selected and primed for identification using 16S ribosomal RNA (rRNA) sequencing technique. The AxyPrep Multisource Genomic DNA Miniprep Kit was used to isolate DNA as described by the manufacturer’s manual. The amount of DNA extracted was electrophoresed on 0.8% agarose gel, and the results obtained were compared with a 1-kb ladder.
PCR amplification and sequencing of 16S rRNA gene from B2 isolate
Polymerase chain reaction (PCR) was performed in a Thermal Cycler. The reaction mixture used consisted of 20 ng of genomic DNA, 2.5 U/50 μl of Taq DNA polymerase, and 5 μl of 10× Taq buffer (100 mM Tris-HCl, 500 mM KCl at pH 8.3), and additionally 200 μM dNTP, 10 pmol each of universal primers (forward primer 27F 5′AGAGTTTGATCCTGGCTCAG3′ and reverse primer 1492R5′TACGGTTACCTTGTTACGACTT3′), and 2.0 mM MgCl2. Amplification process denaturation of the extracted genomic DNA was followed by the annealing of primers at 50 °C for 30 s and extension at 72 °C for 1 and 15 min. Five microliters (5 μl) of the amplified product was analyzed by submarine agarose gel (1.2%) electrophoresis with ethidium bromide at 130 V for 30 min followed by visualization under a Gel Doc/UV transilluminator. This was later gel purified using the Qiagen gel extraction kit and thereafter sequenced with 100 ng/μl of 16S rRNA.
Construction and analysis of RB isolate phylogenetic tree
The 16S rRNA sequences were compared and aligned with sequences deposited in the National Center for Bioinformatics (NCBI) GenBank data base using Basic local Alignment Search Tool (BLAST) (Altschul 1997). The sequences were aligned in CLUSTAL X program, and a phylogenetic tree was constructed by the neighbor-joining method program. The 16S rRNA sequences of bacterial isolate B2 were used for constructing the phylogenetic tree.
Isolation and purification of bioherbicidal substance from RB isolate B2 by HPLC method
Thirty-eight grams per liter (38 g/l) of Mueller Hinton broth was prepared in Milli-Q, beef infusion, casein acid hydrolysate, and starch. This was thereafter sterilized before use as the extraction culture at 30 °C. Isolate B2 was then used to inoculate 25 × 2-l flasks each containing 400 ml of the broth and incubated with a rotary shaker at 200 rpm for 72 h. The broth medium was later centrifuged at 120 rpm for 15 min and the supernatant extracted with ethyl acetate to form an organic layer that was later separated using a separating funnel. The resulting filtrate was dried with sodium sulfate and evaporated at 40 °C using a rotary evaporator to obtain the crude product.
Similarly, the crude extract was defatted in a solution of brine (10 ml), methanol (10 ml), and hexane (20 ml) and stirred for 15 min after which the methanol-water layer was extracted with ethyl acetate. The resulting organic layer was dried with sodium sulfate, evaporated at 40 °C on rotary evaporator, and subjected to flash chromatography. The purified and separated fractions were later tested for herbicidal activity while the fraction with the highest degree of herbicidal activity was analyzed by high-performance liquid chromatography (HPLC) using a C-18 column. Elution was done with acetonitrile/water (90/10 v/v) at a flow rate of 1 ml/min, and detection was done at λ254 while the preparative HPLC was at a flow rate of 3 ml/min (Dueñas et al. 2012).
The seeds of the target weeds (E. crus-galli and A. hybridus) were subjected to viability by water, treated with 15% sodium hypochlorite for 20 min, and rinsed with distilled water (Kordali et al. 2007).
Two layers of filter paper were placed at the base of each clean petri dish (9 cm in diameter) and moistened with 10 ml of distilled water. Fifteen viable seeds of E. crus-galli and A. hybridus were placed on the filter paper, separately for pre-emergence test. Then 2.5, 5, 10, and 15 mg/l of the crude and pure extracts respectively were dropped on the filter paper (Whatman No. 1) according to Kordali et al. (2008). The petri dishes were closed with parafilm to prevent escape of volatile compounds, and incubated at 23 ± 2 °C under 12 h of fluorescent light and relative humidity of 80%. The percentage germination and seedling length were evaluated in a completely randomized setup.
Six soil fungi comprising Aspergillus flavus Link, Fusarium pallidoroseum (Wollenw.) R.F. Castañeda, P. Oliva, Fresneda & N. Rodr., Aspergillus niger Tiegh, Rhizopus stolonifer (Ehrenb.) Stalpers & Schipper, Saccharomyces cerevisiae, and Fusarium oxysporum E.F. Sm. & Swingle were obtained from the culture repository of the Microbiology Department of NSPRI. The test was carried out by growing each fungal species on potato-dextrose-agar (20 ml) amended with 10 and 100 μg/ml of each of the purified fractions respectively. The plates (five per fungal species) were then inoculated with two 7-day-old colony discs and incubated at 25 °C for 15 days. The antifungal effect of the pure fractions isolated from B2 was evaluated by calculating the percentage of linear growth inhibition as 100(y − x)/y, where y = mean colony diameter of toxin-free cultures and x = mean colony diameter of toxin-containing cultures.
Data were analyzed using SPSS software 21. The null hypothesis of equality of mean effect was tested using the two-way ANOVA table at P = 0.05, and means of significant treatments were separated using Duncan’s multiple range tests. Also, the partial eta squared was used to indicate how much of the total variation in the response was accounted for by the factors and their interactions.
Isolation and preliminary screening of rhizopheric bacteria
Characterization and identification of herbicidal active isolate B2
Isolation and purification of herbicidal principle from B2
Herbicidal assessment of fraction 4 on seed germination and seedling performance of target weeds
Inhibitory effects of the purified fraction on seed germination and seedling growth of Amaranthus hybridus and Echinochloa crus-galli
Concentration (mg l−1)
Amaranthus hybridus L.
Echinochloa crus-galli (L.) Beauv.
8.00 ± 0.16de
14.00 ± 0.14c
21.30 ± 0.31e
12.00 ± 0.87c
21.00 ± 0.33b
48.40 ± 0.28b
5.00 ± 0.89e
8.00 ± 0.23d
16.40 ± 1.02f
9.00 ± 0.20cd
18.00 ± 1.00bc
36.20 ± 0.6c
0.00 ± 0.00f
0.00 ± 0.00e
0.00 ± 0.00g
0.00 ± 0.00f
0.00 ± 0.00f
0.00 ± 0.00g
0.00 ± 0.00f
0.00 ± 0.00f
0.00 ± 0.00g
0.00 ± 0.00f
0.00 ± 0.00f
0.00 ± 0.00g
35.20 ± 1.44b
19.30 ± 0.52b
25.30 ± 0.87d
58.90 ± 0.67a
27.20 ± 0.82a
52.50 ± 1.20a
Sensitivity of six soil fungi to the purified fraction from B2
2.67 ± 0.115b
7.53 ± 0.252a
2.4 ± 0.265b
18.77 ± 0.635c
3.5 ± 0.1c
17.5 ± 0.0e
6.3 ± 0.173e
12.83 ± 0.058d
5.7 ± 0.173d
11.97 ± 0.462f
1.6 ± 0.0a
8.87 ± 0.058b
Rhizospheric bacteria from different farm crops across various locations in some parts of Nigeria were randomly investigated for their herbicidal activity, and eight of these isolates from a population of other soil microorganisms were observed to be deleterious to target leaves of A. hybridus and E. crus-galli showing different degrees of necrosis. The isolates with observed necrotic activity may qualify as deleterious rhizospheric bacteria (DBR) as remarked by McPhail et al. (2010). Further investigation involving biochemical, molecular (16S rRNA sequencing technique), and morphological characterization proved that the rhizopheric isolate (B2) with the best necrotic activity per time was P. aeruginosa (Sacchi et al. 2002). This rhizospheric bacterial species was equally implicated in related works by Sessitsch et al. (2004) and McPhail et al. (2010). The reason for the various degrees of necrotic activity observed for the rhizospheric bacterial isolates is unclear; it could be assumed to be due to the genetic disposition of the isolates or a combination of other ecological forces such as local moisture and temperature. Pseudomonas species have been demonstrated in literature as a promising DRB with potential for the biological control of weeds (Kennedy et al. 1991; Flores-Vargas and O’Hara 2006; Kremer and Kennedy 1996; Zermane et al. 2007; Caldwell et al. 2012).
Consequent upon the observed result, P. aeruginosa was processed for the isolation of the active compound(s) that may have been responsible for the necrotic activity. The fraction that was observed to show the highest necrotic activity was later tested on the seed germination and seedling growth of pigweeds and barnyard grass. Complete seed germination and seedling growth inhibition were noted at concentrations of 10 and 15 mg/l, respectively, which may be the lethal dose. The hypersensitivity pattern of the P. aeruginosa-derived fraction 4 noticed during the experimentation suggests an antibiosis type of antagonism (Kaewchai et al. 2009). This confirmed that the fraction 4 derivative of P. aeruginosa was inherently chemically configured for herbicidal activity. While further study is required at characterizing the chemical nature of fraction 4, identifying the role of functional groups as well as molecular weights potentiating herbicidal activity, and developing a potent bioherbicidal formulation (Al-Hinai et al. 2010; Yang et al. 2014). Adetunji and Oloke (2013) and Mejri et al. (2013) have formulated a cell suspension of DRB and pasta granules as effective bioherbicides which precludes the use of pure chemical fractions. Moreover, Pseudomonas fluorescence BRG100 isolated from the rhizosphere of green foxtail in the Brooks of Alberta was reportedly used in controlling green foxtail weeds (Caldwell et al. 2012). Additionally, HPLC application in the processing of derivable crude and its purification was also employed by Zhang et al. (2013) for the isolation as well as elucidation of 4-hydroxy-3-methoxycinnamic acid coupled with two indole derivatives from the fungus Pythium aphanidermatum. The extracted fraction was observed to be non-effective against other non-target soil fungi which further validated its suitability as an environmentally safe bioherbicide (Evidente and Motta 2001).
While the variable phenotypes of the bacterium isolated from the rhizosphere of farm crops was properly identified using molecular technique, their test against pigweed and barnyard grass using leaf necrotic assay showed positivity that validates them as a potentially suitable alternative to mitigating the evidential repercussions of chemical herbicide utility. Furthermore, their test specifically for the non-target effect on soil beneficial fungi underscored the potency of the fraction derived from P. aeruginosa as environmentally safe, target-specific, effective, and cheap and having the potential for commercial formulation.
The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, and The World Academy of Science (TWAS), Italy, for providing the necessary facilities and opportunity to carry out this research. Special thanks to Mr. Rajul Tomar and the whole staff of Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh, India for their contribution to the molecular aspect of this work. Also, I like to appreciate Dr. Adejumo Isaac and Miss Onikanni Olayinka for their input in the statistical analysis.
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